In the first funding cycle, Project 2 was successful in elucidating the nature of OA changes in mice carrying genetic mutations for key fibrillar collagens, including types IX and XI collagen. In the absence of type IX collagen, we learned that complete erosion of joint cartilage occurs with concomitant changes in animal behavior, cartilage biomechanics and chemistry. For Project 2, we take the finding of pro-inflammatory cytokines in the serum of OA patients, as well as our mouse models of OA, and turn towards developing a targeted intervention appropriate for OA based on delivering anti-inflammatory compounds to the joint. Antiinflammatory drugs that attenuate IL-1 and TNFa activity have therapeutic potential for OA but require high protein doses and cause significant side effects when administered via intravenous or subcutaneous injection for inflammatory disease. Strategies that utilize low protein doses and provide for sustained release have great potential to achieve value in the clinic as a treatment for OA. In Project 2, we propose to develop and evaluate the utility of an in situ forming, intra-articular 'drug 'depot' that can provide for local and sustained delivery of anti-inflammatory protein drugs for the treatment of OA. We have previously constructed thermally responsive drug depots the drugs, IL-1 receptor antagonist (IL1Ra) or soluble TNF receptor (sTNFRII), conjugated to a thermally responsive peptide. We have shown that these thermally responsive peptide tags, composed from elastin sequences called ELPs, spontaneously form a depot upon injection into the joint space that provide for a 25-fold increase in the half-life of the administered protein and 75% reduction in peak serum exposure. In Aim 1, we propose studies to evaluate the following for both ELP-IL1Ra and ELP-sTNFRII: (a) in vitro bioactivity against cytokines in primary synoviocytes; (b) in vivo biodistribution following delivery to the rat knee joint; (c) in vitro immunotoxicity; and (d) in vivo efficacy in mediating inflammatory joint disease caused by overexpression of IL-1 and/or LPS injection in the joint space. In Aim 2, we propose to evaluate the disease-modifying effects of ELP-IL1 Ra and ELP-sTNFRII in a joint instability model of OA with the following measures: (a) gross and histological joint appearances; (b) synovial fluid and serum biomarkers (through Core B); and (c) parameters of gait and pain perception. We hypothesize that thermally responsive ELPs conjugated to these anti-inflammatory drugs will contribute to long drug half-lives in the joint space while retaining bioactivity, reducing serum drug exposure and modifying disease in these pre-clinical models of OA. The results of this 5-year project are expected to advance a novel drug depot strategy to easily deliver drugs to the joint, advancing the application of disease-modifying drugs with significant systemic side effects for the treatment of OA.